PROJECT SUMMARY Malaria affects over half the world's population and causes nearly 450,000 deaths every year, mainly in sub- Saharan African children. Rapid development of parasite resistance to frontline antimalarials and lack of an effective, durable vaccine are the most formidable barriers to achieving malaria control and elimination. Host cell invasion by Plasmodium is essential to establish an infection and cause disease. Therefore neutralizing parasites before they enter host cells can have an enormous impact in reducing morbidity and mortality. For this reason proteins on the surface of merozoites (responsible for initiating the disease process) and sporozoites (infectious stage) are being evaluated as potential vaccine targets. However, even the most clinically advanced vaccine RTS,S, targeting the sporozoites, showed limited efficacy. The overall goal of this proposal is to develop a novel vectored gene therapy approach for malaria prophylaxis by targeting a universal pathway required for host cell invasion. This builds on our studies demonstrating a crucial role for the interaction of two Plasmodium proteins, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2), in junction formation between the merozoite and red blood cell (RBC), providing an anchor to facilitate parasite entry. Interestingly, RON2 is secreted onto the host cell and acts as a receptor for AMA1 present on the parasite surface. This is a unique example in host-pathogen interactions where both ligand and receptor are provided by the parasite to mediate successful host cell entry. A 47-amino acid conserved region of RON2 (termed RON2L) is necessary and sufficient to bind a similarly conserved hydrophobic pocket in AMA1. This points to an evolutionarily preserved ?lock-and-key? mechanism designed to prevent changes. Importantly, we demonstrated that RON2L competes for binding to AMA1 and efficiently neutralizes Plasmodium merozoites before they can enter RBCs and recent data suggests that this pathway is also used by P. falciparum sporozoites to enter hepatocytes. We reasoned that targeting this interaction can be an effective antimalarial strategy as it would make it harder for the parasites to develop resistance. To do this, we will develop an adeno-associated virus (AAV)-mediated gene therapy to endogenously express the entry inhibitor RON2L. Preliminary data demonstrated that the entry inhibitor expressed by AAV transduction is produced as a correctly folded, stable fusion protein and potently neutralized P. falciparum merozoites in vitro. We will use an in vivo rodent malaria model and a P. falciparum humanized mouse model to evaluate protection against infection and disease. Successful outcomes of our aims will provide a novel malaria prophylaxis approach by self-targeting a key step used by malaria parasites to enter host cells.